Sayak Banerjee, Amity University Kolkata
In this current spread of outbreaks, specifically with the emergence of more transmissible variants, frequent rapid testing for COVID-19 is a vital requirement. For that purpose, qRT-PCR (quantitative reverse-transcriptase-polymerase chain reaction), the gold standard COVID-19 diagnostic test, is employed these days. This method generally entails specialized equipment, a centralized laboratory facility, and long runtime of several hours. Consequently, it takes the testing a minimum of around one to two days.
A research team from the University of California, Berkley, has professed to develop a diagnostic test that is a lot easier and faster to use than qRT-PCR. The team is led by Jennifer Doudna, who shared the 2020 Nobel Prize in Chemistry for inventing CRISPR-Cas9 genome editing. She said that their work combines two different types of CRISPR enzymes for creating an assay. This assay can identify small amounts of viral RNA in less than an hour. It is said to pick up almost 30 copies of the virus per microliter of liquid, which is more than qRT-PCR. However, the new method has not reached the stage where it matches the sensitivity of qRT-PCR.
Employing an Amplification-free technique:
CRISPR-based assays typically require an initial amplification of the viral RNA for the clear visibility of the detection signal. This signal involves the discharge of a fluorescent molecule that glows under blue light. This initial step could increase the sensitivity of the test to a comparable level of qRT-PCR. Nevertheless, it makes the test more difficult to perform outside a laboratory. The initial amplification step results in billions of copies of viral RNA. It might lead to an increased probability of cross-contamination across patient samples. Whereas, the new method enhances the fluorescence signal, thus eradicating the principal source of cross-contamination.
The scientists named the amplification-free technique as Fast Integrated Nuclease Detection in Tandem (FIND-IT). They said that even though their work was impacting COVID-19, it could apply to many other infectious diseases. In this assay, they used a CRISPR enzyme, Cas13, for the viral RNA detection and a Cas protein, Csm6, to amplify the fluorescence signal. The major role of Cas13 is to cut the RNA, but at very small amounts of target RNA present, it can take hours. To counter this, Csm6 was used to amplify the effect of Cas13.
They engineered an activator molecule to bind to Csm6 and stimulate it to incise and release a bright fluorescent molecule from a piece of RNA. Generally, the activator molecule tends to get degraded by Csm6, and only a limited amount of fluorescent signal is generated. On chemical modification, the activator is protected from degradation and triggers the Csm6 to repeatedly cleave and release fluorescent molecules connected to RNA. Subsequently, the sensitivity becomes 100 times better than the original activator. Additionally, Cas13 results in the production of numerous active enzymes that can cut even more fluorescent reporters.
Significance of Cas13-Cas9 approach:
The team introduced an improved combination of guide RNAs which facilitated more sensitive identification of the viral RNA by Cas13. They were able to determine up to 31 copies per microliter COVID-19 RNA in less than 20 minutes. This technique involving the Cas13 plus Csm6 enzyme approach is carried out in a single reaction at 37 degrees Celsius. On the other hand, any other diagnostic test necessitates multiple steps or higher temperature heating.
From a sensitivity viewpoint, although they had to jump a ten thousandfold gap, they are successful about a thousandfold. They are convinced that the gap is not impossible to cover. Doudna wanted to make a rapid test, in which people can quickly know whether they are infected or not. This approach opens the door to various prospects of faster and simpler tests that can potentially reach higher sensitivities.
Also read: Genetic Predisposition – How Addictions Affect our Genes
Reference:
- Liu, T. Y., Knott, G. J., Smock, D. C. J., Desmarais, J. J., Son, S., Bhuiya, A., Jakhanwal, S., Prywes, N., Agrawal, S., Díaz de León Derby, M., Switz, N. A., Armstrong, M., Harris, A. R., Charles, E. J., Thornton, B. W., Fozouni, P., Shu, J., Stephens, S. I., Kumar, G. R., … Doudna, J. A. (2021). Accelerated RNA detection using tandem CRISPR nucleases. Nature Chemical Biology. https://doi.org/10.1038/s41589-021-00842-2
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Author info:
Sayak Banerjee is a 3rd-year Biotechnology Engineering Student with a great interest in Immunology and Molecular genetics. He is a creative scientific writer in Bioxone with an inclination towards gaining knowledge regarding vast sections of Biotechnology and emphasizing himself in various wet lab skills.
Publications:
- https://bioxone.in/news/worldnews/car-t-cells-scientists-discover-on-off-switches-for-cell-immunotherapy/
- https://bioxone.in/news/worldnews/neutrophil-derived-nanovesicles-a-novel-drug-delivery-system/
- https://bioxone.in/news/worldnews/pig-to-human-heart-transplantation-a-solution-to-the-rarity-of-donor-organs/
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